Summary

Clinical laboratory data on the blood of captive porpoises has been collected from animals being maintained for research at the Navy Marine Bioscience Facility. Tests were conducted over a period of five years on a variety of animals and under differing conditions. This data expands similar information previously reported, and provides some basic normal values for assistance in clinical evaluation.

Introduction

In order to provide comprehensive medical care for porpoises maintained for purposes of research, it has been necessary to determine normal values for various diagnostic tests. Hematology and blood chemistry have been among the most important parameters for clinical evaluation. This paper presents methods and results of laboratory procedures performed on the blood of clinically normal porpoises maintained at the Navy Marine Bioscience Facility.

(From the Marine Bioscience Facility, Naval Undersea Warfare Center, Point Mugu, California, where Dr. Ridgway is Research Veterinarian (Physiology), Dr. Simpson is Research Veterinarian (Clinical), and Mrs. Patton is Chief Medical Laboratory Technologist. We thank F. 0. Wood, Jr. for reading this manuscript.)

The environment among the animals varied, and the environment of individual animals would vary from time to time. Some porpoises were being trained, others were not, some were held in the lagoon adjacent to the Facility and some were confined to artificial pools, while others were maintained in ocean pens. The findings represent data from animals living in varied and changing environments. The animals in this study had been in captivity for six months to five years. Several previous papers (1, 2, 3, 49, 56, 6) have dealt with hematology and blood chemistry of cetaceans; the present work attempts to expand these findings in terms of increased samplings per animal, additional types of laboratory tests, and larger animal numbers and species. The information presented here should be of value to the increasing number of veterinary practitioners charged with the responsibility of maintaining the health of captive cetaceans, as well as to researchers concerned with the extremely interesting physiology of these aquatic mammals.

Materials and Methods

Four species of porpoises were used in this study, the Atlantic bottlenose porpoise (Tursiops truncatus), the Pacific bottlenose porpoise (Tursiops gilli) the Pacific white-striped porpoise (Lagenorhyncus obliquidens), and the Dall porpoise (Phocoenoides dalli). The animals were kept in several types of enclosures and would occasionally be moved from one to another. These enclosures included lagoon pens, concrete tanks, a wooden tank, and floating pens in the open ocean. There was no filtration in the concrete tanks; water quality was maintained by continuous water flow. The wooden tank had a sand and gravel filtration system, and copper sulfate (CuS04) was added. No chemical treatment was used in the other holding facilities and water osmolarity ranged from 852 to 918 mOsm. Air temperature varied from 57 to 71°F while water temperature ranged from 51 to 72°F. Being close to the ocean, considerable fog is experienced throughout the year. Rainfall averages 9.99 inches per year. There are a total of six enclosures, three of which are covered by domes. This is in addition to the lagoon and the ocean pens. The dome-covered enclosures have no artificial climate control, and thus temperature within these structures will vary with ambient temperature.

Blood samples were taken periodically from each of the animals at intervals ranging from one week to several months. No animal was bled less than three times, and some were bled as many as 35 times in the course of this study. Site of venipuncture in most cases was the ventral aspect of the tail flukes, but occasionally the veins which form part of the counter-current vascular system found in the extremities of cetaceans. A 1-1/2-inch, 20-gauge needle was used for venipuncture. Porpoise red blood cells are more fragile than those of dog or man. To minimize hemolysis in blood collection and handling, we employ disposable plastic syringes and test tubes. When glass syringes or test tubes must be used, they are treated with a silicone solution. In addition, the blood is usually centrifuged at low speeds (1,000 to 1,500 RPM) to separate serum or plasma from the cells. Porpoise blood has a prolonged clotting time, unless contaminated by sea water, and does not form a very firm clot in a test tube. Since serum enzyme and potassium levels may change if the serum is allowed to stand on the cells, we centrifuge the blood within one hour after it has been collected whether or not a clot has formed. A fibrin clot may later form in the serum and this is removed. For those samples requiring an anticoagulant, sodium heparin or lipo-heparin was employed.

In preparation for taking samples, the animals were removed from the tank or enclosure on stretchers and placed on a padded table. Water was periodically sprayed over the animal to keep it moist and to maintain normal body temperature. Samples were usually collected before the porpoise was fed. Only porpoises kept in captivity for a period of six months or more are represented in data presented here.

A variety of laboratory methods were used during these studies in order to select the most economical and least complicated procedures consistent with accuracy. An annotated list of techniques follows: (Method of choice is listed first).

• Glucose (1) Folin-Wu (copper reduction) 7,8; (2) Auto Analyzer@; (3) Glucose oxidase (enzvme method) 6,7,8.
• Bun (Blood Urea Nitrogen). (1) Auto Analyzer@; (2) Urograph+++, (7); (3) Chaney-Marbach method (Urease 8 and Berthelot reaction)
• Cholesterol (1) Auto Analyzer@; (2) Modified Carr-Dexter method 7; (3) Modified Ferro-Ham method+.
• Protein Bound Iodine (1) Method of Barker, Humphrey, and Soley (modified dry ash method)
• Sodium and Potassium (1) Flame Photometry*.
• Chloride (1) Buchler-Cotlove Chloridometer++++, (2) Schale and Schale method
• Calcium (1) Auto Analyzer;@ (2) Modified Ferro-Ham method ++(3) Flame Photometer**
• SGOT (Serum Glutamic Oxaloacetic Transaminase). (1) Auto Analyzer@; (2) Trans-ac+++; (3) Amador and Wacker method9; (4) Reitman-Frankel method⁷

@Technicion SMA-12,on SMA-12, Technicon Corp.,Ardsley, N.Y.

*Instrumentation Laboratory, Inc., Watertown, Mass.

+HarlecoCo., Philadelphia, Pa.

++Chem Stat, Inglewood, Calif.

+++General Diagnostics Div-Warner Chilcott, Morris Plains.N.J.

++++Buchler InstrumentsIncFort Lee, N.J.

**Coleman Instruments, Inc., Maywood,

• SGPT (Serum Glutamic Pyruvic Transaminase) (1) Reitman-Frankel method⁷ ; (2) Amador and Wacker (modified)⁹.
• LDH (Lactic Dehydrogenase). (1) Auto Analyzer@ ; (2) Wroblewski and LaDue method^7,8; (3) Amador and Wacker method¹⁰ (4) Amador, Bernstein, and Benotti method¹¹ (5) Berger-Broida method⁷
• Cell Size (1) Unstained slide method; (2) suspension method. Both of these methods are used with the Haden-Hausser Erythrocytometer.
• Hemoglobin (1) Hemoglobin converted to cyanmethemoglobin and read photo electrically; (2) Hemoglobin converted to acid hematin and read by the Haden-Hausser method.
• Hematocrit (1) Microhematocrit; (2) Macro-hematocrit.
• Red Blood Cells (1) Coulter counter*; (2) UnopetteÒ precalibrated automatic Pipet using 0.85% NaCL as the diluent (3) Thoma pipet--Havem's solution or "Gower's solution as the diluent
• White Blood Cells (1) UnoPetteÒ Precalibrated automatic pipet using 1% acetic acid; (2) Thoma pipet--N/10 HCl or 2% acetic acid as the diluent (the former is preferred it using the Haden-Hausser hemoglobin method).

®UnoPette is a registered trademark of Becton,
Dickinson and Co., Rutherford, N.J.
Coulter Electronics, Inc., Hialeah, Florida

• Differential This is a rather straightforward procedure. We have used Wright's staining procedure and Quik-Stain**.
• Osmolality (1) Measuring the freezing point depression by means of an osmometer xx,

Discussion

The data presented in Tables 1-5 have been accumulated over a period of five years, A strict quality control program is carried out in our laboratory as well as in the other laboratories where some of our work was performed. But, as with most laboratory work, there will be some variation in results from a single specimen if it is run simultaneously by separate technicians, or if several methods are used to extract the same data. Thus, in interpreting the information presented here we acknowledge the fact that variations among animals, and in the same animal over a period of time, are a function of differences in the mechanics of laboratory tests, as well as changes in the animal. This is, we believe, a situation that must be acknowledged by all practitioners who evaluate laboratory findings. Values of laboratory procedures performed while animals were obviously ill were not included.

** Laboratory Consultants, LTD, Van Nuys, Calif.
xx The Fiske Osmometer, Fiske Associates, Inc., Oxbridge, Mass.

In our quality control program standards and control serums produced by the following laboratories were employed primarily: (1) Warner-Chilcott Diagnostics Div., Morris Plains, N.J. (2) Dade Reagents, Inc., Miami, Fla. (3) Hyland Laboratories, Los Angeles, Calif.

In our experience porpoises usually, but not always, manifest a strong leucocytic response to infections. Animals with bacterial pneumonias, erysipelas, gastrointestinal infections, and urinary tract infections have manifested white blood counts (WBC) as high as 25,000 to 40,000.

An interesting feature of red cell parameters in these animals is the relationship of packed cell volume and red cell numbers. In the canine an estimation of RBC/cmm can be made by dividing the PCV by six. In the porpoises studied this maneuver will result in an estimated RBC/cmm generally much higher than the RBC count obtained by chamber enumeration. This indicates that the RBC of the porpoise must have a greater volume than the canine RBC. This cannot be accounted greater volume than the canine RBC. for by increased diameter since the diameter of the porpoise RBC is in the range of the human and the canine RBC. The MCV (mean corpuscular volume) of the porpoise RBC is ordinarily over 100 cubic microns while in the canine the maximum normal is 77 cubic microns 12, and in the adult human (female) the 13 maximum is reported to be 98 cubic microns. The cow is reported to have a maximum normal MCV of 54 cubic microns 13 which is interesting in that many of the anatomic features of the porpoise are similar to those of the bovine. The large MCV of the porpoise would suggest an adaptation to the respiratory requirements incumbent with an aquatic environment.

Blood sugar in the porpoise normally ranges from 100 to 150 mg%. One porpoise that we have studied consistently runs a blood sugar of over 200 mg% without glycosuria or clinical signs of diabetes. Another porpoise at a nearby oceanarium developed high blood sugar levels14 accompanied by clinical signs of diabetes It reportedly responded satisfactorily to therapy directed towards the diabetic symptoms. We have not as yet found the enzyme levels (transaminases, lactic dehydrogenase) to be of significant diagnostic value, but probably with continued accumulation and evaluation of this data, they will prove clinically useful. It was noted in one Pacific white-striped porpoise that had gone off feed, and failed to show other behavioral or clinical signs of illness, that the SGOT level rose to 610 units and then dropped to normal levels when customary eating habits returned. This suggests the possibility that SGOT measures non­specific cellular catabolism in porpoises. The high percentage of eosinophils in the differential count on white blood cells has been reported previously^2,3,5 as has the relatively large value for BUN.^4,5. No explanation is presently apparent for this situation, but the high BUN may have something to do with osmoregulation, and the eosinophils may result from some physiological adaptation to the marine environment and food supply.

In reviewing the accumulated data on individual animals some conclusions were sought regarding relationship of total WBC to stress (e.g. newly caught animals). No consistent pattern could be established, such as high WBC with apparent stress. One explanation for this is the fact that these animals vary markedly in regard to temperament. Some are quite placid, others demonstrate evident hostility, while others display what appears to be nervousness when handled. We also have not been able to detect marked differences in laboratory findings based on sex. There are, however, apparent species variations. The Dall porpoise (P. dalli) is reportedly the deepest diving animal of the three species in this study, and runs a relatively high hemoglobin level.¹⁵ The assumption is that this is an adaptation to deep diving and fast swimming. Other correlative studies have indicated this deep diving and fast swimming adaptation relative to the oxygen dissociation curve16Lagenorhyncus RBC and hemoglobin average higher than those of the Tursiops and we assume that because of this Tursiops, in its natural environment, is the shallowest diver of the three species. The cell diameter of the white-striped porpoise and Dall porpoise is less than that of the Atlantic bottlenose porpoise. There are interesting but as yet not completely understood implications to this in view of the fact that the MCV of Tursiops runs higher than that of the other species, yet the hemoglobin and packed cell volume tend to be lower. Although Lagenorhyncus and Phocoenoides have a smaller cell size, the RBC in these species runs around 5.5 million whereas this value in Tursiops is usually around 4 million.

Serum osmolarity in our porpoises averaged about 339 mOsm, a value about 40 to 50 m0sm higher than values found in horses, cattle, and man, but only slightly higher than values reported for pigs and sheep.¹³ From Table III it can be seen that serum sodium levels in porpoises average about 153 mEQ/L and chlorides average about 110 mEQ/L, thus these ions occur in porpoises serum at levels roughly 10 mEQ/L higher than in man but in the same general range as pigs, sheep, and dogs. The serum potassium values from Table III range slightly lower than those reported for man. This is interesting in view of the fact that fish which porpoises subsist on contain large amounts of potassium and levels of this element in marine fishes are reported to be about five times higher than are sodium levels.¹⁷

Cholesterol levels vary significantly between Lagenorhyncus and Tursiops. In the latter species this constituent commonly runs over 200 Mg% while in Lagenorhyncus the levels are generally under 200 Mg%. In humans, blood levels of cholesterol are only partially influenced by dietary intake,¹⁸ and we would assume that this is also the case in cetaceans. Biosynthesis of cholesterol appears to be the major factor in blood cholesterol levels. Of particular interest is the finding that while humans and porpoises have similar blood cholesterol levels, the former subsist on a diet relatively high in saturated fat, while porpoises live on a fish diet which is high in unsaturated fats.

Introduction to Tables

Several comments are necessary to augment and clarify the following tables:

1. Abbreviations for animal species are:

• Tursiops truncatus--Tt
• Tursiops gilli--Tg
• Lagenorhynchus obliquidens-Lo
• Phocoenoides dalli--Pd

2. In the hematologic tables, nucleated RBC are not included in that their occurrence is rare on blood smears.

3. Animals Tt 76 and 77 had ovariohysterectomies performed on them and the blood data was derived after these surgeries. Animal Tt 70 had been used for deep dive studies and was being worked in the open ocean during most of this period.

4. There were inadequate single animal recordings of blood serum inorganic phosphorous, thus we pooled data with the following results:

Editors’ Note: The tables are not very clear in the original pdf but have been included here for your reference.

References

1. Morimoto, H. M., Takata, and J. Sudzuki. Tohuko Journal of Experimental Medicine, 2 (1921).
2. Andersen, S. Nord. Vet. Med., 18:51-56 (1966).
3. Medway, W. and J. R. Geraci. Am.J. of Physiol., 207: 1367-1370 (1964).
4. Medway, W. and J. R. Geraci, Am. J. of Physiol., 209: 169-172 (1965),
5. Ridgway, S. J.A.V.M.A., 147:1077-1085 (1965).
6. Eichelberger, L., E. S. Fetcher, E.M.K. Geiling, and B. J. Vos. J. Biol. Chem., 133:145-152 (1940).
7. Frankel and Reitman. Gradwohl's Clinical Laboratorv Methods and Diagnoses, 6th Ed., C. V. Mosby Co., St. Louis, 1963.
8. Henry, R. J. Clinical Chemistry-Principles and Technics, Harper and Row, New York, 1964.
9. Amador, E. and W. E. C. Waker. Clin. Chem., 8:343 (1962).
10. Amador, E., L. E. Dorfman and W.E.C. Wacker. Clin. Chem., 9:391 (1963).
11. Amador, F., H. Reinstein, and N. Benotti, Am. J. Clin. Path., 44:62 (1965).
12. Schalm. Veterinary Hematology, 2nd Ed., Lea and Febiger, Philadelphia, 1965.
13. Altman, P. L. and D. S. Dittmer. Blood and Other Body Fluids, lst Ed., Federation of American Societies for Experimental Biology, Washington, D. C., 1961.
14. Kenney, David. Del Mar, California, personal communication.
15. Ridgway, S. H. and D. G. Johnston. Science, 151:456-458 (1966).
16. Horvath, S. M., H. Chiodi, S. H. Ridgway, and S. Azar. J. Comp. Physiol. and Biochem., In Press (1967).
17. Thurston, C. E. J. Am. Dietet. Assoc., 34(4): 396-400 (1958).
18. Harper, H. A. Review of Physiological Chemistry, 10th Ed, Lange Medical Publications, Los Altos, Calif, (1965).